Aspirin is one of the most promising agents for the chemoprevention of human cancer. The clinical usefulness of aspirin is partially limited by its side effects. The overall aim of this research proposal is to test the hypothesis that transcription factor AP-1 is the molecular target for the anti-tumor promotion effect of aspirin in JB6 cells and an AP-1- luciferase transgenic mouse model. This mechanism may also be applicable to other systems, in addition to or instead of the "prostaglandin H synthase inhibition" mechanism, for the anti-tumor promotion activity of aspirin. Such knowledge will help to develop better agents with fewer side effects for effective chemoprevention of human cancer. The specific aims are: Aim 1 - To determine whether inhibition of tumor promoter-induced cell transformation by aspirin is dependent on the inhibition of AP-1 activity; Aim 2 - To determine whether prostaglandin H synthase is involved in the inhibiition of tumor promoter-induced AP-1 activity and cell transformation by aspirin or salicylic acid; Aim 3 - To determine whether inhibition of AP-1 acitivity and transformation of aspirin is through a mitogen- activated protein kinase (MAP kinase) pathway or through the elevation of intracellular H plus concentration; Aim 4 - To determine whether aspirin inhibits AP-1 activity and tumor promotion in vivo under the two-stage carcinogenesis conditions in an AP-1 luciferase transgenic mouse model. The strategy for Specific Aim 1 is to establish a cell culture model for studying anti-tumor promotion activity of aspirin by using the JB6 cell system. We will then test whether AP-1 activity is inhibited in the same anti-tmor promotion conditions. We will also test the activity of anti-tumor promotion and inhibition of AP-1 by other nonsteroidal anti-inflammatory drugs (NSAIDs). For Specific Air 2, we will use indomethacin and other NSAIDs, which do not inhibit AP-1 activity and are potent prostaglandin H synthase inhibitors to test whether they inhibit or promote JB6 cell transformation. We will alos test the direct effect of prostaglandins on AP-1 activity and cell transformation. For Specific Aim 3, we will use antibodies specific for the phosphorylated tyrosine 204 of Erk1 or Erk2, and Ser63 or Ser73 of c-Jun to study the phosphorylated Erk1 or Erk2, c-Jun (Ser63/Ser73) and JNK activity. We will measure the effect of aspirin on intracellular pH by a pH dependent fluorescence dye and study the role of intrascellular pH on AP-1 activity and cell transformation by using a H plus pump inhibitor, diethylstilbestrol and overexpression of ATPase PMA gene and its mutants in JB6 cells. For Specific Aim 4, we will use an AP-1 luciferase transgenic mouse model to investigate whether aspirin inhibits AP-1 activity in vivo under the two-stage carcinogenesis conditions. Furthermore, we will test whether the same dosage of aspirin that inhibits tumor promoter-induced AP-1 activity also inhibits tumor promotion in the transgenic mice.